Down syndrome (DS) is a major cause of mental retardation and congenital heart disease affecting the welfare of more than 300,000 individuals and their families in the USA alone. In addition, DS syndrome carries an increased risk of leukemia, congenital gut disease, defects of the immune system, and an Alzheimer-like dementia. Little is known about the molecular events that cause the DS features or of the genes that determine the early embryonic stage whose program is disrupted. The ultimate goal of the research described in this proposal is to define the genes and their functions responsible for the phenotypic features of DS. The specific aims of this proposal are designed to identify the chromosome 21 genes responsible for DS congenital heart disease (DS-CHD) and for holoprosencephaly (HP) or abnormal forebrain cleavage, due to monosomy for chromosome 21 (HP-21). Molecular markers for chromosome 21 consensus regions that are likely to contain the genes for congenital heart disease and holoprosencephaly have been defined. The specific aims are: 1) To define the limits of the DS-CHD region by molecular analyses of DS-CHD cell lines, using DNA probes not yet used to analyze this region including 26 new markers mapping in this region, and by the genetic analysis of autosomal dominant endocardial cushion defect; and for 21-HP, by molecular analyses of cell lines from 21-HP deletion and 40 spontaneous HP cases with normal chromosomes; 2) To define Yeast artificial chromosomes and cosmids within these regions; 3) To define genes that map in these regions and are expressed in both the human embryo and the fetal heart (DS-CHD) or brain (21-HP). To do this, human embryonic and fetal heart and brain cDNA libraries will be constructed and genes present in the YACs and cosmids will be defined using exon amplification and hybrid selection techniques, from 21 microclones, and from cDNAs with developmental motifs; 4) To characterize DS-CHD and 21-HP cDNAs by establishing map location, DNA sequence, expression in human (normal and DS) and mouse (normal and trisomy 16) embryonic and fetal tissues, protein studies, and ultimately, transgenic mice. These studies will define genes involved in the developmental programming of the human embryonic brain and heart that map on chromosome 21 and may be in part responsible for DS congenital heart disease and mental retardation.